Lactoferrin receptor protein

ABSTRACT

An isolated and purified lactoferrin receptor protein is isolated and purified from bacterial pathogens, including Moraxella and Neisseria, and has a molecular weight of between about 70,000 and about 90,000, as determined by SDS-PAGE. Such lactoferrin receptor protein may be provided in combination with a lactoferrin receptor protein from the bacterial pathogen of a molecular weight of about 100,000 to about 105,000 daltons. The lactoferrin receptor protein may be produced by providing a solubilized membrane preparation from the bacterial pathogen containing lactoferrin receptor proteins, non-lactoferrin receptor proteins and other contaminants, complexing the lactoferrin receptor proteins with lactoferrin and purifying the resulting complexes substantially free from the non-lactoferrin receptor proteins and the other contaminants, and separating the novel lactoferrin receptor protein from the complexes. The lactoferrin receptor protein is useful in diagnostic applications and immunogenic compositions, particularly for in vivo administration to a host to confer protection against disease caused by a bacterial pathogen that produces the lactoferrin receptor protein or produces a protein capable of inducing antibodies in a host specifically reactive with the lactoferrin receptor protein.

This application is a division of application Ser. No. 08/552,232 filedNov. 2, 1995 (now U.S. Pat. No. 6,048,539).

FIELD OF THE INVENTION

The present invention relates to the field of immunology and isparticularly concerned with a novel lactoferrin receptor proteinisolated and purified from bacterial pathogens and methods ofproduction, and uses thereof.

BACKGROUND TO THE INVENTION

Most living organisms require a continuous supply of iron to maintaingrowth and thus have evolved efficient mechanisms for acquisition ofiron under conditions of limitation (ref. 1—Throughout this application,various references are referred to in parenthesis to more fully describethe state of the art to which this invention pertains. Fullbibliographic information for each citation is found at the end of thespecification, immediately preceding the claims. The disclosures ofthese references are hereby incorporated by reference into the presentdisclosure) A common mechanism found in many bacterial species involvesthe synthesis and secretion of small iron-chelating molecules,siderophores, which complex with iron and are subsequently bound andinternalized via specific receptors at the bacterial surface (ref. 2).This mechanism is effective in a wide variety of environments and isoften found in bacterial species that are present in a variety ofecological niches.

The vertebrate host provides an iron-restricted environment to potentialbacterial pathogens, largely due to the sequestration of extracellulariron by the host iron-binding glycoproteins, namely transferrin (Tf) andlactoferrin (Lf). Although a siderophore-mediated mechanism should beeffective in this setting, some bacterial species have evolvedalternative mechanisms of iron acquisition that are adapted to theirparticular host. Thus, some members of the Pasteurellaceae andNeisseriaceae are capable of specifically binding and effectivelyacquiring iron from the host Tf and in some cases, Lf. This is mediatedby receptors present at the bacterial surface whose expression isinduced by restricting the level of available iron in the medium.

Receptors that are specific for Tf from the host (ref. 3) have beenidentified in a variety of important human and veterinary pathogens aswell as some commensal species (Table 1). To date, Tf receptors haveonly been identified in bacterial species within the Pasteurellaceae andNeisseriaceae. In most species the Tf receptor has been shown to consistof two proteins, Tf-binding protein I (Tbp1) and Tf-binding protein 2(Tbp2). The genes encoding these proteins have been cloned fromNeisseria meningitidis (ref. 13), N. gonorrhoeae (refs. 14 and 15),Haemophilus influenzae (ref. 16) and Actinobacillus pleuropneumoniae(refs. 17 and 18). The predicted amino acid sequences of Tbp2 proteinsreveal homology to the lipoprotein signal peptidase recognition sitesuggesting that it is lipid-modified and retains an association with theouter membrane via its lipid tail. Lipidation of Tbp2 has been confirmedby labelling (refs. 13 and 19) and evidence is accumulating that Tbp2 islargely surface exposed (refs. 20 and 21). Isogenic mutants deficient inthe production of Tbp2 demonstrate severely limited ability to utilizetransferrin as a sole iron source during in vitro growth studies,suggesting Tbp2 plays an important, albeit not essential role in ironacquisition (refs. 14, 16, 22).

Based on its homology with other TonB-dependant outer membrane proteinsTbp1 receptor proteins are believed to have several regions which spanthe outer membrane (ref. 23) (see ref. 35 for a topological model thatcan be applied to Tbp1). Similarly, based on the results obtained withthe FepA receptor (ref. 25), Tbp1 is thought to act as a gated porewhich allows the passage of iron from the transferrin and lactoferrinmolecules, which are themselves not internalized, to the periplasm whereit is bound by the ferric iron-binding protein, FbpA. Two additionalproteins FbpB and FbpC are believed to be involved in the transport ofiron across the cytoplasmic membrane.

The transport of iron across the outer membrane is believed to occur ina TonB-dependent manner, in that amino acid sequences referred as the“TonB box”, located in a number of TonB-dependent outer membranereceptor proteins have also been identified in Tbp1. The inability toutilize human transferrin following insertional inactivation of the H.influenzae TonB homologue clearly supports this theory. In addition,mutants in which the Tbp1 protein has been insertionally inactivated areunable to utilize transferrin as a sole iron source, supporting itsessential role in iron acquisition from transferrin.

Bacterial lactoferrin receptors have only been described for humanpathogens in the Neisseriaceae, and were thought to consist of a singleprotein, Lbp1. Amino acid sequence analysis of the Lbp1 protein showshigh homology to Tbp1 (refs. 26, 35, 36), and isogenic mutant analysisdeficient in Lbp1 suggests an essential role of Lbp1 in iron acquisition(refs. 24 and 28). Recent genetic evidence suggests that similar to thetbpBA operonic organization, an open reading frame is locatedimmediately upstream of the lbpA structural gene which may encode a Tbp2homologue, Lbp2 (ref. 35).

Properties of lactoferrin receptor proteins of bacterial pathogensindicate that these proteins have utility in diagnosis of andvaccination against diseases caused by such bacterial pathogens thatproduce lactoferrin receptor proteins or proteins capable of raisingantibodies specifically reactive with lactoferrin receptor proteins.

It would be advantageous to provide purified lactoferrin receptorproteins (and methods of purification thereof) for use as antigens,immunogenic preparations, including vaccines, carriers for otherantigens and immunogens and the generation of diagnostic reagents.

SUMMARY OF THE INVENTION

The present invention is directed towards the provision of purified andisolated lactoferrin receptor protein from a bacterial pathogen having amolecular mass of about 70,000 and about 90,000 daltons wherein themolecular mass is determined by sodium dodecyl sulphate polyacrylamidegel electrophoresis (SDS-PAGE).

In accordance with one aspect of the invention, there is provided alactoferrin receptor protein isolated and purified from a bacterialpathogen and having an apparent molecular mass of about 70,000 to about90,000 daltons as determined by SDS-PAGE.

The bacterial pathogens from which the lactoferrin receptor protein maybe isolated and purified include Neisseria meningitidis, Neisseriagonorrhoeae, Moraxella catarrhalis, Moraxella bovis and Moraxellalacunata. The about 70 to about 90 kDa lactoferrin receptor protein isat least about 70 wt % pure, preferably at least about 90 wt % pure, andmay be in the form of an aqueous solution thereof.

The lactoferrin receptor protein, sometimes referred to herein by thedesignation “Lbp2”, may be provided in a form substantially free from alactoferrin receptor protein having a molecular weight of about 100,000to about 105,000 daltons, as determined by SDS-PAGE, sometimes referredto herein by the designation “Lbp1”.

Alternatively, the novel lactoferrin receptor protein provided hereinmay comprise a mixture of the lactoferrin receptor proteins. In anotheraspect of the present invention, therefore, there is provided acomposition comprising a first lactoferrin receptor protein (Lbp1)having a molecular weight of about 100,000 to about 105,000 and a secondlactoferrin receptor protein having a molecular weight of between about70,000 and about 90,000 daltons, wherein the molecular weights aredetermined by SDS-PAGE and the lactoferrin receptor proteins areisolated and purified from a bacterial pathogen, which may be any of thepathogens mentioned above.

Such composition may be at least about 70% pure by weight, preferably atleast about 90% pure by weight. Such composition also may be providedsubstantially free from transferrin receptor proteins of the pathogen.

The present invention also provides an immunogenic compositioncomprising an immunoeffective amount of an active component, which maybe the novel lactoferrin receptor protein, provided herein alone ortogether with Lbp1 along with a pharmaceutically acceptable carriertherefor. The immunogenic composition may be formulated as a vaccine forin vivo administration to a host to confer protection against diseasescaused by a bacterial pathogen which produces a lactoferrin receptorprotein having a molecular weight of about 70,000 to about 90,000daltons, or that produces a protein capable of inducing antibodies inthe host specifically reactive with the lactoferrin receptor protein.The immunogenic composition may be formulated as a microparticlecapsule, ISCOM or liposome preparation. The immunogenic composition maybe used in combination with a targeting molecule for delivery tospecific cells of the immune system or to mucosal surfaces. Sometargeting molecules include strain B12 and fragments of bacterialtoxins, as described in WO 92/17167 (Biotech Australia Pty. Ltd.), andmonoclonal antibodies, as described in U.S. Pat. No. 5,194,254 (Barberet al). The immunogenic compositions of the invention (includingvaccines) may further comprise at least one other immunogenic orimmunostimulating material and the immunostimulating material may be atleast one adjuvant. Suitable adjuvants for use in the present inventioninclude, (but are not limited to) aluminum phosphate, aluminumhydroxide, QS21, Quil A, calcium phosphate, calcium hydroxide, zinchydroxide, a glycolipid analog, an octadecyl ester of an amino acid, amuramyl dipeptide and a lipoprotein. Advantageous combinations ofadjuvants are described in copending U.S. patent application Ser. No.08/261,194 filed Jun. 16, 1994, assigned to the assignee hereof and thedisclosure of which is incorporated herein by reference. The immunogeniccomposition may be substantially free from transferrin receptor proteinof the pathogen.

The invention further includes an antibody specific for the novellactoferrin receptor protein provided herein producible by immunizing ahost with an immunogenic composition as provided herein.

In a further aspect of the invention, there is provided a method ofgenerating an immune response in a host, comprising administeringthereto an immuno-effective amount of the immunogenic composition asprovided herein. The immune response may be a humoral or a cell-mediatedimmune response. Hosts in which protection against disease may beconferred include primates including humans.

The immune response which is generated may provide protection to thehost against disease caused by a bacterial pathogen that produces alactoferrin receptor protein having a molecular weight of between about70,000 and about 90,000 daltons or that produces a protein capable ofinducing antibodies in the host specifically reactive with thelactoferrin receptor protein.

The present invention provides, in an additional aspect thereof, amethod for producing a vaccine comprising administering the immunogeniccomposition provided herein to a test host to determine an amount and afrequency of administration of the lactoferrin receptor protein toconfer protection against disease caused by a bacterial pathogen thatproduces the lactoferrin receptor protein having a molecular weightbetween about 70,000 and about 90,000 or a protein capable of inducingantibodies in a host specifically reactive with the lactoferrin receptorprotein; and formulating the lactoferrin receptor protein in a formsuitable for administration to a treated host in accordance with saiddetermined amount and frequency of administration. The treated host maybe a human.

A further aspect of the invention provides a method of determining thepresence in a sample, of antibodies specifically reactive with alactoferrin receptor protein of a bacterial pathogen having a molecularweight of between about 70,000 and about 90,000 daltons, comprising thesteps of:

(a) contacting the sample with the lactoferrin receptor protein asprovided herein to produce complexes comprising the lactoferrin receptorprotein and any said antibodies present in the sample specificallyreactive therewith; and

(b) determining production of the complexes.

In a further aspect of the invention, there is provided a method ofdetermining the presence, in a sample, of a lactoferrin receptor proteinof a bacterial pathogen and having a molecular weight of between about70,000 and about 90,000 daltons, comprising the steps of:

(a) immunizing a subject with the lactoferrin receptor protein asprovided herein, to produce antibodies specific for the lactoferrinreceptor protein;

(b) contacting the sample with the antibodies to produce complexescomprising any outer membrane protein present in the sample and saidouter membrane protein specific antibodies; and

(c) determining production of the complexes.

A further aspect of the invention provides a diagnostic kit fordetermining the presence of antibodies in a sample specifically reactivewith the lactoferrin receptor protein of a bacterial pathogen and havinga molecular weight of between about 70,000 and about 90,000 daltons,comprising:

(a) the lactoferrin receptor protein as provided herein;

(b) means for contacting the lactoferrin receptor protein with thesample to produce complexes comprising the lactoferrin receptor proteinand any said antibodies present in the sample; and

(c) means for determining production of the complexes.

The invention also provides a diagnostic kit for detecting the presence,in a sample, of a lactoferrin receptor protein of a bacterial pathogenand having a molecular weight of between about 70,000 and about 90,000,comprising:

(a) an antibody specific for the novel lactoferrin receptor protein asprovided herein;

(b) means for contacting the antibody with the sample to produce acomplex comprising the lactoferrin receptor protein and lactoferrinreceptor protein-specific antibody; and

(c) means for determining production of the complex.

In an additional aspect, the present invention provides a method ofproducing a vaccine, comprising administering the immunogeniccomposition provided herein to a test host to determine an amount and afrequency of administration to confer protection against disease causedby a bacterial pathogen that produces a lactoferrin receptor proteinhaving a molecular weight of between about 70,000 and about 90,000daltons or a protein capable of inducing antibodies in a test hostspecifically reactive with the lactoferrin receptor protein; andformulating the immunogenic composition in a form suitable foradministration to a treated host, which may be a human, in accordancewith the determined amount and frequency of administration.

The present invention additionally provides a method of producingantibodies specific for a lactoferrin receptor protein having amolecular weight of between about 70,000 and about 90,000 daltons,comprising:

(a) administering the lactoferrin receptor protein provided herein to atleast one mouse to produce at least one immunized mouse;

(b) removing B-lymphocytes from the at least one immunized mouse;

(c) fusing the B-lymphocytes from the at least one immunized mouse withmyeloma cells, thereby producing hybridomas;

(d) cloning the hybridomas;

(e) selecting clones which produce anti-lactoferrin receptor proteinantibody;

(f) culturing the anti-lactoferrin receptor protein antibody-producingclones; and then

(g) isolating anti-lactoferrin receptor protein antibodies from thecultures.

The present invention further includes procedures for isolating andpurifying the novel lactoferrin receptor protein from a bacterialpathogen, either in a form free from Lbp1 or in a form of a purifiedmixture with Lbp1.

Accordingly, in another aspect of the invention, there is provided amethod of producing a lactoferrin receptor protein from a bacterialpathogen, comprising the steps of:

(a) providing a solubilized membrane preparation from the bacterialpathogen containing lactoferrin receptor proteins, non-lactoferrinreceptor proteins and other contaminants,

(b) complexing the lactoferrin receptor proteins with lactoferrin andpurifying the resulting complexes substantially free fromnon-lactoferrin receptor proteins and the other contaminants, and

(c) separating from the complexes a lactoferrin receptor protein havinga molecular weight of between about 70,000 and about 90,000 daltons.

This isolation and purification procedure may be carried out in anydesired manner. The lactoferrin receptor proteins may further comprise asecond lactoferrin receptor protein having a molecular weight of about100,000 to about 105,000 daltons (i.e. lbp1).

In one embodiment, step (b) is effected by complexing the twolactoferrin receptor proteins with lactoferrin and separating thecomplexes from the membrane preparation, and step (c) produces apurified mixture of the two lactoferrin receptor proteins.

In another embodiment, the non-lactoferric receptor protein includetransferrin receptor proteins of the pathogen, step (b) is effected byremoving the second lactoferrin receptor protein (Lbp1) and thetransferrin receptor proteins (Tbp1 and Tbp2) from the solubilizedmembrane preparation to provide a depleted membrane preparation,complexing the first-mentioned lactoferrin receptor protein withlactoferrin in the depleted membrane preparation and removing thecomplex so formed from the depleted membrane preparation.

In forming the complexes with the transferrin receptor protein(s), thetransferrin may be coupled to a carrier molecule, which may be aSepharose, such as cyanogen bromide activated CH-Sepharose. In oneembodiment, the lactoferrin may be conjugated to biotin and theSepharose in streptavidin-Sepharose to couple the lactoferrin tostreptavidin.

In this application, the term “lactoferrin receptor protein” is used todefine a family of lactoferrin receptor proteins of a variety ofpathogens having a molecular mass of between about 70,000 and 90 daltonsand includes proteins having variations in their amino acid sequencesincluding those naturally occurring in various strains of bacterialpathogens. In this application, a first protein or peptide is a“functional analog” of a second protein or peptide if the first proteinis immunologically related to and/or has the same function as the secondprotein or peptide. The functional analog may be, for example, afragment of the protein or a substitution, addition or deletion mutantthereof. The invention also extends to such functional analogs.

Advantages of the present invention include:

a method for isolating purified about 70,000 to about 90,000 daltonslactoferrin receptor protein of a bacterial pathogen that produces thelactoferrin receptor protein;

an isolated and purified lactoferrin receptor protein of a bacterialpathogen having a molecular weight of about 70,000 to about 90,000daltons as determined by SDS-PAGE; and

diagnostic kits and immunological reagents for specific identificationof bacterial pathogens and hosts infected thereby.

BRIEF DESCRIPTION OF THE FIGURES

The present invention will be further understood from the followingdescription with reference to the Figures, in which:

FIG. 1 shows an SDS-PAGE analysis of transferrin isolated bindingproteins and lactoferrin binding proteins isolated from bacterialpathogens;

FIG. 2 shows the specific identification by immunoblot analysis oftransferrin binding proteins and lactoferrin binding proteins isolatedfrom bacterial pathogens;

FIG. 3 shows an SDS-PAGE analysis of lactoferrin binding proteins Lbp1and Lbp2 isolated from Moraxella catarrhalis; and

FIG. 4 shows the specificity of transferrin binding protein for bindingtransferrin and lactoferrin binding protein for binding lactoferrin.

GENERAL DESCRIPTION OF INVENTION

The present invention provides a novel lactoferrin receptor proteinwhich is isolated and purified from a pathogen and has a molecularweight of between 70,000 and 90,000 daltons as determined by SDS-PAGEand herein referred to sometime as Lbp2. There is some heterogenicity insize among the Lbp2 protein from different species, as seen in FIG. 1.The novel Lbp2 protein may be isolated from a variety of bacterialpathogens, including Neisseria meningitidis, Neisseria gonorrhoeae,Moraxella catarrhalis, Moraxella bovis and Moraxella lacunata.

The Lbp2 protein may be isolated using affinity purification procedures.In U.S. Pat. No. 5,141,743, there is described an affinity purificationprocedure which results in the isolation of two transferrin receptorproteins, Tbp1 and Tbp2, from a variety of bacterial pathogens,including Moraxella catarrhalis (FIG. 1, lane 2), Neisseria meningitidis(lanes 8, 11) and M. bovis (lane 5). Using conditions selected tominimize isolation of contaminating proteins, a single host-specificlactoferrin receptor protein, Lbp1, is isolated from the same speciesand strains (lanes 3, 6, 9 and 12).

Such affinity purification procedure is effected using transferrin orlactoferrin, as the case may be, which normally is complexed to asuitable matrix material, such as Sepharose, for example, though linkagevia cyanogen bromide activated CH-Sepharose or biotin-LC-hydrazide,which is subsequently joined to Streptavidin Sepharose. The transferrin-or lactoferrin-matrix conjugate is contacted with a solubilized membranepreparation from the bacterial pathogen, following removal of solublematerial, under pH and salt conditions, generally high levels of saltand pH, which causes the receptor protein to define the transferrin orlactoferrin, as the case may be. The resulting solid complexes areseparated from the liquid phase and the respective proteins may berecovered by elution of the solid complexes with a gradient ofchaotropic agents, such as guanidine hydrochloride.

To obtain the Lbp2 alone from the solubilized membrane preparation, theLbp1 and transferrin receptor proteins first are removed and theincubation with the lactoferrin-matrix conjugate is repeated under lowsalt and pH conditions, resulting in solid complex formation. Followingremoval of the solid complexes, the Lbp2 protein can be removed byelution from the solid complex.

To obtain from the solubilized membrane preparation the Lbpt2 inadmixture with Lbpt1, the solubilized membrane preparation may becontacted with the transferrin-matrix conjugate under the high salt andpH conditions favouring non-binding the Lbpt2 and then separating thesolid complexes formed, thereby separating the Lbpt1 and Lbpt2 proteinsfrom the transferrin receptor proteins.

Following affinity isolation of Tbp1+2, Lbp1 and Lbp1+2, the sampleswere subjected to SDS-PAGE and were subsequently electroblotted andanalyzed for reactivity against various antisera, as seen in FIG. 3.Following affinity isolation of Lbp2, the purified preparations wereimmobilized on a nitrocellulose membrane and tested for binding activityin the solid-phase binding assay (FIG. 3).

In additional embodiments of the present invention, the Lbp2 protein asprovided herein may be used as a carrier molecule to prepare chimericmolecules and conjugate vaccines (including glycoconjugates) againstpathogenic bacteria, including encapsulated bacteria. Thus, for example,glycoconjugates of the present invention may be used to conferprotection against disease and infection caused by any bacteria havingpolysaccharide antigens including lipooligosaccharides (LOS) and PRP.Such bacterial pathogens may include, for example, Haemophilusinfluenzae, Streptococcus pneumoniae, Escherichia coli, Neisseriameningitidis, Salmonella typhi, Streptococcus mutans, Cryptococcusneoformans, Klebsiella, Staphylococcus aureus and Pseudomonasaeruginosa. Particular antigens which can be conjugated to Lbp2 proteinand methods to achieve such conjugations are described in published PCTapplication WO 94/12641, the disclosure of which is hereby incorporatedby reference thereto.

In another embodiment, the carrier function of Lbp2 protein may be used,for example, to induce immunity toward abnormal polysaccharides of tumorcells, or to produce anti-tumor antibodies that can be conjugated tochemotherapeutic or bioactive agents.

The invention extends to lactoferrin binding proteins as provided hereinfor use as an active pharmaceutical substance, particularly as an activeingredient in a vaccine against disease caused by infection with apathogen.

It is clearly apparent to one skilled in the art, that the variousembodiments of the present invention have many applications in thefields of vaccination, diagnosis, treatment of bacterial infections andthe generation of immunological reagents. A further non-limitingdiscussion of such uses is further presented below.

1. Vaccine Preparation and Use

Immunogenic compositions, suitable to be used as vaccines, may beprepared from the lactoferrin receptor protein, as well as analogs andfragments thereof, as disclosed herein. The vaccine elicits an immuneresponse which produces antibodies, including anti-lactoferrin receptorantibodies and antibodies that are opsonizing or bactericidal. Shouldthe vaccinated subject be challenged by a bacterial pathogen thatproduce a lactoferrin receptor protein having a molecular weight ofbetween about 70,000 and about 90,000 daltons, the antibodies bind tothe lactoferrin receptor and thereby prevent access of the bacterialpathogen to an iron source which is required for viability. Furthermore,opsonizing or bactericidal anti-lactoferrin receptor protein antibodiesmay also provide protection by alternative mechanisms.

Immunogenic compositions including vaccines may be prepared asinjectables, as liquid solutions or emulsions. The lactoferrin receptorprotein may be mixed with pharmaceutically acceptable excipients whichare compatible therewith. Such excipients may include, water, saline,dextrose, glycerol, ethanol, and combinations thereof. The immunogeniccompositions and vaccines may further contain auxiliary substances, suchas wetting or emulsifying agents, pH buffering agents, or adjuvants toenhance the effectiveness thereof. Immunogenic compositions and vaccinesmay be administered parenterally, by injection subcutaneously orintramuscularly. Alternatively, the immunogenic compositions formedaccording to the present invention, may be formulated and delivered in amanner to evoke an immune response at mucosal surfaces. Thus, theimmunogenic composition may be administered to mucosal surfaces by, forexample, the nasal or oral (intragastric) routes. Alternatively, othermodes of administration including suppositories and oral formulationsmay be desirable. For suppositories, binders and carriers may include,for example, polyalkalene glycols or triglycerides. Oral formulationsmay include normally employed incipients such as, for example,pharmaceutical grades of saccharine, cellulose and magnesium carbonate.These compositions can take the form of solutions, suspensions, tablets,pills, capsules, sustained release formulations or powders and containabout 1 to 95% of the lactoferrin receptor protein. The immunogenicpreparations and vaccines are administered in a manner compatible withthe dosage formulation, and in such amount as will be therapeuticallyeffective, protective and immunogenic. The quantity to be administereddepends on the subject to be treated, including, for example, thecapacity of the individual's immune system to synthesize antibodies, andif needed, to produce a cell-mediated immune response. Precise amountsof active ingredient required to be administered depend on the judgmentof the practitioner. However, suitable dosage ranges are readilydeterminable by one skilled in the art and may be of the order ofmicrograms of the lactoferrin receptor protein. Suitable regimes forinitial administration and booster doses are also variable, but mayinclude an initial administration followed by subsequentadministrations. The dosage may also depend on the route ofadministration and will vary according to the size of the host.

The concentration of the lactoferrin receptor protein Lbp2, with orwithout the corresponding Lbp1, in an immunogenic composition accordingto the invention is in general about 1 to 95%. A vaccine which containsantigenic material of only one pathogen is a monovalent vaccine.Vaccines which contain antigenic material of several pathogens arecombined vaccines and also belong to the present invention. Suchcombined vaccines contain, for example, material from various pathogensor from various strains of the same pathogen, or from combinations ofvarious pathogens. In particular applications, it may be desirable toprovide mixtures comprising immunologically distinct lactoferrin bindingproteins, including immunologically-distinct Lbp2.

Immunogenicity can be significantly improved if the antigens areco-administered with adjuvants, commonly used as 0.05 to 0.1 percentsolution in phosphate-buffered saline. Adjuvants enhance theimmunogenicity of an antigen but are not necessarily immunogenicthemselves. Adjuvants may act by retaining the antigen locally near thesite of administration to produce a depot effect facilitating a slow,sustained release of antigen to cells of the immune system. Adjuvantscan also attract cells of the immune system to an antigen depot andstimulate such cells to elicit immune responses.

Immunostimulatory agents or adjuvants have been used for many years toimprove the host immune responses to, for example, vaccines. Intrinsicadjuvants, such as lipopolysaccharides, normally are the components ofthe killed or attenuated bacteria used as vaccines. Extrinsic adjuvantsare immunomodulators which are typically non-covalently linked toantigens and are formulated to enhance the host immune responses. Thus,adjuvants have been identified that enhance the immune response toantigens delivered parenterally. Some of these adjuvants are toxic,however, and can cause undesirable side-effects, making them unsuitablefor use in humans and many animals. Indeed, only aluminum hydroxide andaluminum phosphate (collectively commonly referred to as alum) areroutinely used as adjuvants in human and veterinary vaccines. Theefficacy of alum in increasing antibody responses to diphtheria andtetanus toxoids is well established and a HBsAg vaccine has beenadjuvanted with alum. While the usefulness of alum is well establishedfor some applications, it has limitations. For example, alum isineffective for influenza vaccination and inconsistently elicits a cellmediated immune response. The antibodies elicited by alum-adjuvantedantigens are mainly of the IgG1 isotype in the mouse, which may not beoptimal for protection by some vaccinal agents.

A wide range of extrinsic adjuvants can provoke potent immune responsesto antigens. These include saponins complexed to membrane proteinantigens (immune stimulating complexes), pluronic polymers with mineraloil, killed mycobacteria in mineral oil, Freund's complete adjuvant,bacterial products, such as muramyl dipeptide (MDP) andlipopolysaccharide (LPS), as well as lipid A, and liposomes.

To efficiently induce humoral immune responses (HIR) and cell-mediatedimmunity (CMI), immunogens are often emulsified in adjuvants. Manyadjuvants are toxic, inducing granulomas, acute and chronicinflammations (Freund's complete adjuvant, FCA), cytolysis (saponins andPluronic polymers) and pyrogenicity, arthritis and anterior uveitis (LPSand MDP). Although FCA is an excellent adjuvant and widely used inresearch, it is not licensed for use in human or veterinary vaccinesbecause of its toxicity.

Desirable characteristics of ideal adjuvants include:

(1) lack of toxicity;

(2) ability to stimulate a long-lasting immune response;

(3) simplicity of manufacture and stability in long-term storage;

(4) ability to elicit both CMI and HIR to antigens administered byvarious routes, if required;

(5) synergy with other adjuvants;

(6) capability of selectively interacting with populations of antigenpresenting cells (APC);

(7) ability to specifically elicit appropriate T_(H)1 or T_(H)2cell-specific immune responses; and

(8) ability to selectively increase appropriate antibody isotype levels(for example, IgA) against antigens.

U.S. Pat. No. 4,855,283 granted to Lockhoff et al on Aug. 8, 1989 whichis incorporated herein by reference thereto teaches glycolipid analoguesincluding N-glycosylamides, N-glycosylureas and N-glycosylcarbamates,each of which is substituted in the sugar residue by an amino acid, asimmuno-modulators or adjuvants. Thus, Lockhoff et al. (U.S. Pat. No. 4,855,283 and ref. 29) reported that N-glycolipid analogs displayingstructural similarities to the naturally-occurring glycolipids, such asglycosphingolipids and glycoglycerolipids, are capable of elicitingstrong immune responses in both herpes simplex virus vaccine andpseudorabies virus vaccine. Some glycolipids have been synthesized fromlong chain-alkylamines and fatty acids that are linked directly with thesugars through the anomeric carbon atom, to mimic the functions of thenaturally occurring lipid residues.

U.S. Pat. No. 4,258,029 granted to Moloney, incorporated herein byreference thereto, teaches that octadecyl tyrosine hydrochloride (OTH)functioned as an adjuvant when complexed with tetanus toxoid andformalin inactivated type I, II and III poliomyelitis virus vaccine.Also, Nixon-George et al. (ref. 30), reported that octadecyl esters ofaromatic amino acids complexed with a recombinant hepatitis B surfaceantigen, enhanced the host immune responses against hepatitis B virus.

2. Immunoassays

The lactoferrin receptor protein of the present invention is useful asan immunogen for the generation of anti-lactoferrin receptor proteinantibodies, as an antigen in immunoassays including enzyme-linkedimmunosorbent assays (ELISA), RIAs and other non-enzyme linked antibodybinding assays or procedures known in the art for the detection ofanti-bacterial and anti-Lbp2 antibodies. In ELISA assays, thelactoferrin receptor protein is immobilized onto a selected surface, forexample, a surface capable of binding proteins such as the wells of apolystyrene microtiter plate. After washing to remove incompletelyadsorbed lactoferrin receptor protein, a nonspecific protein, such as asolution of bovine serum albumin (BSA) that is known to be antigenicallyneutral with regard to the test sample, may be bound to the selectedsurface. This allows for blocking of nonspecific adsorption sites on theimmobilizing surface and thus reduces the background caused bynonspecific bindings of antisera onto the surface.

The immobilizing surface is then contacted with a sample, such asclinical or biological materials, to be tested in a manner conducive toimmune complex (antigen/antibody) formation. This may include dilutingthe sample with diluents, such as solutions of BSA, bovine gammaglobulin (BGG) and/or phosphate buffered saline (PBS)/Tween. The sampleis then allowed to incubate for from about 2 to 4 hours, at temperaturessuch as of the order of about 25° to 37° C. Following incubation, thesample-contacted surface is washed to remove non-immunocomplexedmaterial. The washing procedure may include washing with a solution,such as PBS/Tween or a borate buffer. Following formation of specificimmunocomplexes between the test sample and the bound lactoferrinreceptor protein, and subsequent washing, the occurrence, and evenamount, of immunocomplex formation may be determined by subjecting theimmunocomplex to a second antibody having specificity for the firstantibody. If the test sample is of human origin, the second antibody isan antibody having specificity for human immunoglobulins and in generalIgG. To provide detecting means, the second antibody may have anassociated activity such as an enzymatic activity that will generate,for example, a colour development upon incubating with an appropriatechromogenic substrate. Quantification may then be achieved by measuringthe degree of colour generation using, for example, a visible spectraspectrophotometer.

EXAMPLES

The above disclosure generally describes the present invention. A morecomplete understanding can be obtained by reference to the followingspecific Examples. These Examples are described solely for purposes ofillustration and are not intended to limit the scope of the invention.Changes in form and substitution of equivalents are contemplated ascircumstances may suggest or render expedient. Although specific termshave been employed herein, such terms are intended in a descriptivesense and not for purposes of limitations.

Methods of molecular genetics, protein biochemistry, and immunology usedbut not explicitly described in this disclosure and these Examples areamply reported in the scientific literature and are well within theability of those skilled in the art.

Example 1

This Example describes the growth of bacteria.

Neisseria and Moraxella species cells were propagated in liquid BrainHeart Infusion broth (BHI; Gibco Laboratories). Neisseria meningitidisM982 and M986 were obtained from Dr. E. Mackie and Dr. C. E. Frasch,(FDA, Bethesda), whereas P3006, used for PCR amplification of theC-terminal region of the N. meningitidis lbpB gene region, was obtainedfrom Dr. Jan Poolman (University of Amsterdam). The Moraxellacatarrhalis and M. bovis isolates used for these studies have beendescribed previously (refs. 31 and 32).

For binding and affinity isolation experiments, iron-restriction ofbacterial cells was performed as described previously. Growth studieswere performed using plate assays in which iron sources were applied toBrain Heart Infusion (Gibco) agar plates containing 100 μMEtheylenediaminedi(o-hydroxyphenyllacetic) acid (BHI+EDDA) on which asuspension of iron-limited cells had previously been spread (ref. 34).200 μg of each iron-saturated transferrin or lactoferrin was appliedonto the sterile disks prior to application to the plates. The plateswere examined after 24 and 48 hours of incubation for cell growthsurrounding the disks containing the various iron sources.

Example 2

This Example describes the ability of selected bacterial pathogens toutilize various iron sources.

Iron saturated bacteria were spread onto BHI plates containing EDDA (100μM) as described above and discs containing human transferrin (hTf),bovine transferrin (bTf), human lactoferrin (hLf) and bovine lactoferrin(bLf). Human lactoferrin and transferrin, and bovine transferrin usedfor the binding and growth studies was obtained from Sigma, USA. Bovinelactoferrin was obtained from Serva, USA. The bacteria tested were N.meningitidis a human pathogen; N. meningitidis lbpA::Gm which isdeficient in lactoferrin binding protein; N. meningitidis tbpA::Kanwhich is deficient in transferrin binding protein; M. catarrhalis (ahuman pathogen) and M. bovis (a bovine pathogen). Growth surrounding thedisk impregnated with the iron source was the measure of the ability ofthe various bacteria to utilize the iron source. The results are shownin Table 2 and indicate that:

(a) each of the pathogens could utilize transferrin or lactoferrin asthe source of iron and thus express transferrin and lactoferrin receptorproteins;

(b) the human pathogen (N. meningitidis and M. catarrhalis) were onlyable to utilize human transferrin and human lactoferrin;

(c) the bovine pathogen (M. catarrhalis) was only able to utilize bovinetransferrin and bovine lactoferrin; and

(d) deletion of the genes encoding transferrin receptor protein (Tbp1)and lactoferrin receptor protein (Lbp1) from N. meningitidis preventedthe utilization of transferrin and lactoferrin respectively.

Example 3

This Example describes the preparation ofbiotin-LC-hydrazide-lactoferrin derivatives.

Covalent linkage of biotin to lactoferrin was achieved usingbiotin-LC-hydrazide which can be used to couple biotin to carbohydrategroups following mild periodate oxidation. Equal volumes of ice coldhuman lactoferrin (2 mg/ml) and 20 mM sodium periodate dissolved in 0.1M sodium acetate buffer (pH 5.5) to a concentration of 2 mg/ml wereadded together and allowed to react on ice in the dark for 20 minutes.Glycerol was subsequently added to a final concentration of 15 mM toquench the reaction, and the sample was dialyzed overnight againstacetate buffer. Biotin-LC-Hydrazide dissolved in DMSO was added to thedialyzed samples to a final concentration of 5 mM, and the mixture wasincubated at room temperature for two hours with mild agitation, thendialyzed against 20 mM Tris buffer, pH 8.

Example 4

This Example describes the isolation and characterization of transferrinreceptor protein 1 (Tbp1), transferrin receptor protein 2 (Tbp2),lactoferrin receptor protein (Lbp1) and lactoferrin receptor protein 2(Lbp2), by affinity isolation.

Transferrin receptor proteins Tbp1 and Tbp2 and lactoferrin receptorprotein Lbp1 have been affinity isolated previously as described aboveand U.S. Pat. No. 5,141,743 which is incorporated herein by referencethereto and ref. 34. Transferrin-Sepharose and lactoferrin-Sepharosecolumns were prepared from the transferrin and lactoferrin preparationsby coupling a 10 mg/ml solution of transferrin or lactoferrin toactivated Sepharose (Pharmacia, Uppsala, Sweden) according to themanufacturer's instruction for coupling ligands tocyanogenbromide-activated Sepharose (CNBr-Sepharose). The resultingcolumn was washed with 2 column volumes of 50 mM Tris buffer (pH 8.0)containing 6 M guanidine hydrochloride to remove non-covalently boundtransferrin or lactoferrin.

Bacterial cells resuspended from fresh cultures on chocolate plates wereused to inoculate prewarmed Brain Heart Infusion broth containing 100 μMEDDA (ethylenediaminedi(O-hydroxyphenyl)acetic acid) to a starting A₆₀₀of 0.02. The resulting culture was incubated at 37° C. with shaking for16 hours prior to harvest by centrifugation at 9,000×g for 15 minutes.The cells were resuspended to 0.2 gm/ml in 50 mM TrisHCl, pH 8 buffercontaining 50 μg/ml phenylmethylsulfonyl fluoride. The cells were lysedby passing the suspension through a French pressure cell at 16,000lb/in₂ and cell debris was removed by centrifugation at 9,000×g for 15minutes. Crude total membranes were collected by centrifugation at140,000×g for 1 hour and resuspended in 50 mM TrisHCl, pH 8 buffer.

Ten mg of crude membrane protein was diluted in 1 ml of 50 mM Tris-HCl,1 M NaCl, 20 mM EDTA (pH 8.0) buffer containing 0.75% Sarkosyl NL37. Thesolubilized membrane preparation was centrifuged at 10,000 rpm for 10minutes to remove cell debris. The supernatant containing outer membranecomponents was mixed with 100 μl of the transferrin-Sepharose resin orlactoferrin-Sepharose resin and incubated for 1 hour to allow thebinding of transferrin receptor protein or lactoferrin receptor proteinto its respective ligand. The resin was collected by centrifugation at500×g for 10 minutes and resuspended in 50 mM TrisHCl, 1 M NaCl, 10 mMEDTA, 0.75% “SARKOSYL”, pH 8 buffer. The resin was again collected bycentrifugation and washed two more times in the above buffer. After thefinal wash, the resin was resuspended in 20 mls of the above buffer andpoured into 1 cm diameter chromatography column. The packed resin waswashed with an additional 10 mls of 50 mM TrisHCl, 1 M NaCl, 10 mM EDTA,0.5% “SARKOSYL” and the receptor proteins were eluted by application ofa 60 ml gradient of 1 to 3 M guanidine hydrochloride in 50 mM TrisHCl, 1M NaCl, 10 mM EDTA, 0.05% “SARKOSYL”. The fractions containing receptorproteins were pooled and dialyzed against two changes of 3 litres of 50mM TrisHCl, pH 7.5 buffer and one change of phosphate buffered saline(50 mM sodium phosphate, 150 mM NaCl, pH 7.4) and concentrated byultrafiltration. These previously developed affinity methods result inthe isolation of two human transferrin-specific receptor proteins, Tbp1and Tbp2, from a variety of species including the human pathogensMoraxella catarrhalis (FIG. 1, lane 2), and Neisseria meningitides(strain M982, lane 8; strain P3006, lane 11). Similarly, twobovine-specific transferrin receptor proteins are isolated from thebovine pathogen, M. bovis (lane 5). Using these conditions (originallyselected to minimize isolation of contaminating proteins) a singlehost-specific lactoferrin receptor protein (lanes 3, 6, 9 and 12) isisolated from these same species and strains.

For the isolation of mixtures comprising a first lactoferrin receptorprotein having a molecular weight of about 100,000 to about 105,000daltons (Lbp1) and a second lactoferrin receptor protein having amolecular weight of between about 70,000 and about 90,000 (Lbp2)modifications were made to these affinity isolation procedures. Thus,total membranes of iron-starved M. catarrhalis, M. bovis and N.meningitidis were diluted to a protein concentration of approximately 10mg/ml and solubilized in 50 mM Tris pH 6 buffer containing 10 mMEtheylenediaminetetraacetic acid (EDTA), 0.6% Sarkosyl and 0.76 M NaCl,then insoluble matter was removed by centrifugation. To achieve affinityisolation of Lbp 1+2, the sample was diluted tenfold with 50 mm Trisbuffer pH 6, and incubated in the presence of human lactoferrincovalently coupled to CNBr-activated CH-Sepharose orbiotin-LC-hydrazide, for 2 hours at room temperature. For the humanlactoferrin-biotin-LC-hydrazide conjugate, the sample was subsequentlyincubated in the presence of Streptavidin-Sepharose for an additionalhour. For analytical purposes, after centrifugation, the pellets werewashed three times with pH 6, 50 MM Tris buffer containing 0.1 M NaCl,then resuspended in a small volume of Laemmli sample buffer, and boiledfor 5 minutes prior to subjecting the samples to SDS-PAGE, using a 10%acrylamide gel. This modified lactoferrin receptor protein affinityisolation procedure resulted in isolation of a second major protein fromM. catarrhalis (FIG. 1, lane 4), M. bovis (FIG. 1, lane 7) and N.meningitidis (strain M982, FIG. 1, lane 10; strain P3006, FIG. 1, lane13). This protein has a molecular weight of between about 70,000 and90,000 and is shown by an asterisk in FIG. 1. This lactoferrin receptorprotein is herein referred to as binding Lbp2. For preparative isolationof the Lbp1 and Lbp2 proteins, the washed pellets were poured into a 1cm diameter chromatography column and the proteins were eluted byapplication of 2M guanidine hydrochloride.

There is considerable heterogeneity in the size of the Tbp2 proteinisolated from different strains and species of bacteria. (FIG. 1,arrowhead). Although there may be some variation in size amongst theLbp2 proteins from different species, they are much more uniform insize.

To further characterize the lactoferrin receptor protein Lbp2, varioussera were tested for reactivity to the protein. Following affinityisolation of Tbp1+2, Lbp1 and Lbp1+2, the samples were subjected toSDS-PAGE, and were subsequently electroblotted and analyzed forreactivity against various antisera (FIG. 2). The separated proteinswere subsequently electrophoretically transferred onto Nitrocellulosemembranes (Western blot). After blocking the remaining binding siteswith a solution of Tris-buffered saline (TBS) containing 0.5% skim milk,the blots were incubated in the presence of rabbit polyclonal antisera(1:5000) directed towards either the affinity isolated M. catarrhalisTbp1+2 proteins, Lbp1 protein, or towards human lactoferrin. In FIG. 2,Lane 1 contains transferrin receptor proteins Tbp1 and Tbp2 isolatedfrom M. catarrhalis, Lane 2 contains lactoferrin receptor protein Lbp1,Lanes 3 and 4 contain mixtures comprising a first lactoferrin receptorprotein having a molecular weight of about 100,000 to about 105,000daltons (Lbp1) and a second lactoferrin receptor protein having amolecular weight of between about 70,000 and about 90,000 (Lbp2). Themixture in Lane 4 was isolated utilizing lactoferrin biotinylated viaits oligosaccharide side chain (Lane 4). In FIG. 2, Panel A is stainedwith Coomassie brilliant blue. When tested with antisera against Tbp1and Tbp2 (Panel B), reactive bands representing Tbp1 (asterisk) and Tbp2(dot) were observed in the samples from the modified affinity methods(lanes 3 and 4). Although careful inspection of the results clearlyindicates that these are distinct from the major bands isolated with thelactoferrin affinity resin, it is also evident that the modifiedaffinity method results in preparations that contain numerouscontaminating proteins.

Reaction of the electroblotted proteins with antiserum against Lbp1(Panel C) confirmed that the major upper band (open arrowhead) was Lbp1.This result also demonstrated that the second band Lbp2 (closedarrowhead) was not a breakdown product of Lbp1. Antiserum preparedagainst human lactoferrin did not react with the second major band Lbp2(closed arrowhead) but with a minor contaminating band that was moreevident in samples obtained from the modified affinity methods (opendiamond, particularly lane 4).

Although the results in FIGS. 1 and 2 clearly demonstrate that anadditional protein is isolated with the modified affinity methods usinglactoferrin as the ligand, they do not conclusively demonstrate thatthis protein Lbp2 is binding directly to lactoferrin. Therefore apurified preparation of Lbp2, as well as the other receptor proteins wasobtained and binding properties evaluated. To selectively affinitypurify the Lbp1 and Lbp2, the sample was processed under standardconditions for Lbp1 isolation, and allowed to incubate for 1 hour withhuman lactoferrin-Sepharose. After separating the resin bycentrifugation, the supernatant containing the Lbp2 was diluted tenfoldwith 50 mM pH 6 Tris buffer, and incubated for two hours with a secondaliquot of human lactoferrin-Sepharose, and processed as described abovefor Lbp1+2 isolation. The human lactoferrin-Sepharose-bound Lbp1 andLbp2 were eluted using 50 mM Tris buffer, pH 8 containing 3 MGuanidine-HCl and 0.3% Sarkosyl, and dialyzed against 50 mM Tris-HClbuffer, pH 8, then lyophilized.

To selectively purify the Lbp1+2 or Lbp2 alone without contaminating Tbp1+2 present, it was necessary to affinity isolate the Tbpl+2 from thesolubilized membranes, using human transferrin-Sepharose prior tosubjecting the extract to the modified affinity isolation protocol.Using this strategy purified preparations of Tbp1+2 (FIG. 3, lane 1),Lbp1 (lane 2) and Lbp2 (lane 3) were obtained. The purified preparationswere immobilized on a nitrocellulose membrane and subsequently testedfor binding activity in the solid-phase binding assay. The results areshown in FIG. 4 and demonstrated that, similar to Lbp1, Lbp2specifically bound labelled human lactoferrin but not labelled humantransferrin nor labelled bovine lactoferrin.

Example 5

This Example describes the specific binding of lactoferrin andtransferrin by receptor proteins by a solid-phase binding assay.

The selectively eluted Tbp1+2, Lbp1 alone or Lbp2 alone were spottedonto HA-nitrocellulose paper placed into a dot blot apparatus withvacuum apparatus attached. The remaining binding sites on the blot wereblocked with Tris-buffered saline containing 0.5% skim milk. Theindividual wells of the blot were then incubated with horse-radishperoxidase-labelled human lactoferrin (HRP-hLf), human transferrin(HRP-hTf), or bovine lactoferrin (HRP-bLf) in the presence or absence ofexcess unlabelled hLF or hTf.

Example 6

This Example describes the polymerase chain reaction (PCR) amplificationof the N. meningitides P3006 lbpB C-terminal region. An open readingframe presumptively encoding a Tbp2 homologue was identified upstream ofthe lbpA gene in meningococcal strain H44/76 (refs. 22, 35). The targetDNA to be amplified was prepared by growing an overnight liquid cultureof organisms, transferring 1.5 ml to a sterile microfuge tube,centrifuging the sample, and discarding the supernatant. The cell pelletwas resuspended in a small volume of sterile water, boiled for 5minutes, centrifuged and the supernatant was used as template forsubsequent PCR analysis. One primer was designed to match the knownsequence of the lbpB coding region of the C-terminal portion of theH44/76 LbpB (Lbp2) (approximately the last quarter of the codingsequence), and the second primer was designed to match the region whichencodes for the signal peptide region of the LbpA (Lbp1) protein. Theregion was amplified using chromosomal DNA from strain P3006. Thisstrain was shown to possess a functional Lbp2 protein which could beaffinity purified (FIG. 1, lane 13). Annealing temperatures used for theamplification were stringent (greater that 55° C.), and an extensiontime of one minute with standard extension and DNA-melting parameters(72° C. and 94° C. respectively). Commercially available Taq DNApolymerase (Bethesda Research Labs) and a Perkin Elmer DNA ThermalCycler were used for the DNA amplification. The resulting 557 base pairPCR product was cloned directly into the vector pCRII, which allowsdirect cloning of PCR fragments amplified using Taq DNA polymerase. Anisolate was chosen that had the PCR fragment cloned in the oppositeorientation of the pCRII lac promoter region so that any potentiallytoxic Lbp2-fusion proteins would not be produced, and thus decreasingthe likelihood of lbpb gene rearrangements. The DNA was sequenced, andthe derived amino acid sequence was compared to that of known Lbp2 andTbp2 derived amino acid sequences (Table 3). The protein has a number ofregions that showed considerable homology to the Lbp2 and Tbp2 proteinsof the various pathogens. Also there is a stretch of amino acids whichis rich in negatively-charged residues evident in M986 and H44/76 Lbp2s(−50 to −16 and −57 to −16, respectively, Table 4) that was not presentin the P3006 Lbp2.

SUMMARY OF THE DISCLOSURE

In summary of this disclosure, the present invention provides a novellactoferrin receptor protein, which is isolated and purified from asolubilized membrane preparation from a pathogen, having a molecularweight of between about 70,000 and about 90,000 daltons, compositionscomprising the same and procedures for the preparation thereof.Modifications are possible within the scope of this invention.

TABLE 1 Bacterial Species with Transferrin or Lactoferrin Receptors HostBacterial Species Receptor Disease Reference Human Neisseriameningitides Tf, Lf meningitis 4 Areisseria gonorrhoeae Tf, Lf gonorrhea5 Moraxella catarrhalis Tf, Lf otitis media 5 Moraxella lacunata Tf, Lfconjunctivitis this work Haemophilus influenzae Tf meningitis 6 BovineMoraxella bovis Tf, Lf keratoconjunctivitis 7 Pasteurella haemolytica Tfshipping fever 8 Pasteurella multocida Tf haemorrhagic septicemia 9Haemophilus somnus Tf TEME 10  Equine Actinobacillus equuli Tf 7 PorcineActinobacillus pleuropneumoniae Tf pneumonia 11  Avian Haemophilusparagallinarum Tf infectious coryza 12  Haemophilus avium Tf 12 

TABLE 2 Utilization of Lactoferrin and Transferrin Iron for GrowthGrowth of Bacteria utilizing the Protein Source of Iron Bacterial StrainhTf bTf hLf bLf N. meningitidis + − + − N. meningitidis 1bpA::Gm + − − −N. meningitidis tbpA::Kan − − + − N. catarrhalis + − + − M. bovis − +− + +, growth surrounding the disk containing the specified iron source;−, no detectable growth

TABLE 3 Comparative Analysis of Lbp2 and Tbp2 Amino Acid Sequences AminoAcid Amino Acid Species Strain Protein Alignment Position AlignmentPosition Reference N. meningitidis H44/76 Lbp2 GGFYGPQAEELGG (−70 to−58) VVFGAK (−14 to −9) 21 N. meningitidis P3006 Lbp2 ********A**** (−37to −25) ****** (−14 to −9) This work N. meningitidis M986 Lbp2******E*A**** (−77 to −65) ****** (−14 to −9) 36 N. meningitidis M982Tbp2 ******K****** (−52 to −40) ****** (−12 to −7) 37 N. meningitidisB16B6 Tbp2 *****KN*I*M** (−42 to −30) ****** (−12 to −7) 37 N.gonorrhoeae FA19 Tbp2 ******N****** (−52 to −40) ****** (−14 to −9) 38R. influenzae DL63 Tbp2 *A****H*T**** (−51 to −39) ****** (−14 to −9) 39A. pleuropneumoniae H49 Tbp2 ******K***MA* (−35 to −23) A***** (−11 to−6) 40 A. pleuropneumoniae AP205 Tbp2 ******K***MA* (−35 to −23) A*****(−11 to −6) 41 Note: * indicates identical amino acid. The stop signalis the “0” position. This was done since the start of the Lbp2 protein(i.e. the true number of amino acids is not yet known).

TABLE 4 Comparative Analysis of N. meningitidis H44/76, P3006 and M986Lbp2 amino acid sequences Strain Alignment H44/76LKGIRTAEADIPQTGKARYIGTWEARI (−183 to −157) P3006*****************H********* (−161 to −135) H44/76IQWDNHADKKAAKAEFDVDFGEKSISGTLTEKNGVQPAFHIENGVIEGNGFHATARTRDNG (−153 to−93) P3006 **K**Y*-NQG***Q*T****A**L**K*****DTH***Y**K***D******L****E**(−119 to −60) H44/76 STNPPSFKANNLLVT  (−85 to −71) P3006 ****Q****S****G (−53 to −39) H44/76GGFYGPQAEELGGTIFNNDGKSLGITEDTENEAEAEVENEAGVG-------EQLKPEAKPQFGVVFGAKKDNKEVEK* (−70 to 0) P3006********A****N*IDS*R*I----------------------------------------*********MQ***** (−37 to 0) M986******E******I*************G***KV**D*DVDVD*DVDADADV******V******************** (−77 to 0) Note: * indicates identical amino acid. - indicates gaps inthe amino acid alignment. The stop codon is the “0” position, since theentire Lbp2 sequence is not yet known. The M986 Lbp2 sequence was notincluded in all alignments since only limited Lbp2 amino acid sequencewas available. In addition, the M986 lbpb sequence was changed from theoriginal published sequence to add a G at position 1, and a C atposition 15. This allows for a presumed correct reading frame of thelbpB gene and encodes for the highly conserved GGFYG.

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What we claim is:
 1. A method of producing a lactoferrin receptorprotein from a bacterial pathogen selected from the group consisting ofNeisseria meningitdis, Neisseria gonorrhoeae, Moraxella catarrhalis,Moraxella bovis and Moraxella lacunata, comprising the steps of: (a)providing a solubilized membrane preparation from said bacterialpathogen containing lactoferrin receptor proteins, transferrin receptorproteins and other contaminants, said lactoferrin receptor proteinsbeing a first lactoferrin receptor protein having a molecular weight of100,000 to 105,000 daltons, as determined by sodium dodecyl sulphatepolyacrylamide gel electrophoresis (SDS-PAGE), and a second lactoferrinreceptor protein having a molecular weight of between 70,000 and 90,000daltons, as determined by SDS-PAGE, (b) complexing both said first andsecond lactoferrin receptor proteins with lactoferrin and purifying theresulting complexes free from said transferrin receptor proteins andsaid other contaminants to provide purified complexes, and (c)separating from the purified complexes both said first and secondlactoferrin receptor proteins in the form of a mixture.
 2. A method ofproducing a lactoferrin receptor protein from a bacterial pathogenselected from the group consisting of Neisseria menngitidis, Neisseriaaonorrhoeae, Moraxella catarrhalis, Moraxella bovis and Moraxellalacunata, which comprises the steps of: (a) providing a membranepreparation from the bacterial pathogen, (b) solubilizing the membranepreparation to form a solubilized membrane preparation containing aselected lactoferrin receptor protein, transferrin receptor protein andother contaminants, wherein said selected lactoferrin receptor proteinhas a molecular weight of 100,000 to 105,000 daltons, as determined bySDS-PAGE (c) complexing said selected lactoferrin receptor protein withlactoferrin and complexing said transferrin receptor protein withtransferrin and purifying the resulting lactoferrin- andtransferrin-complexes free from each other and said other contaminantsto provide a first purified lactoferrin complex and purified transferrincomplex, thereby removing said selected lactoferrin receptor protein andsaid transferrin receptor proteins from said solubilized membranepreparation to provide a depleted membrane preparation, (d) solubilizingsaid depleted membrane preparation to form a solubilized membranepreparation containing a second lactoferrin receptor protein having amolecular weight of between 70,000 and 90,000 daltons, as determined bySDS-PAGE, (e) complexing said second lactoferrin receptor protein withlactoferrin and removing the complex so formed from said depletedmembrane preparation to provide second lactoferrin complexes, (f)separating from the first lactoferrin complexes said selectedlactoferrin receptor protein, and (g) separating from the secondlactoferrin complexes said second lactoferrin receptor protein.
 3. Themethod of claim 1 or 2 wherein said lactoferrin is coupled to a carriermolecule.
 4. The method of claim 3 wherein the carrier molecule isSepharose.
 5. The method of claim 4 wherein the Sepharose is cyanogenbromide activated CH-Sepharose.
 6. The method of claim 4 wherein thelactoferrin is conjugated to biotin and the Sepharose isstreptavidin-Sepharose to couple the lactoferrin to the Sepharose.